The following invention is directed generally to the therapeutic utilization of white blood cells, a technique for sequestering the white blood cells by causing them to coalesce in a population density greater than they normally occur in nature, and a method for causing an enriched concentration in conjunction with an array of bags oriented in a set that facilitates both the concentration process and a method for preserving the white blood cells.
It is now recognized that placenta/umbilical cord blood (PB) contains large numbers of hematopoietic stem and progenitor cells that endow PB with extraordinary therapeutic capabilities in the reconstitution of bone marrow damaged as a result of inherited diseases, accidents or medical procedures. As in the case of ordinary collection of bone marrow for transplantation, PB contains immune cells potentially capable of mounting specific responses against the recipients of such transplants, but in contrast to adult immunological cells, those in PB display a lower, perhaps much lower tendency to produce damaging immune responses against the recipient. The clinical syndrome produced by the immuno responses of the graft against the recipient""s cells and tissues is designated xe2x80x9cGraft versus Host Diseasexe2x80x9d (GVHD). In the typical clinical situation, the recipient""s own immune response against the graft is abrogated by drugs and irradiation treatments designed to reduce or eliminate the immunological and other hematopoietic cells and thus avoid the host versus graft immune reaction that would cause rejection of the graft. It has been proven that the principal targets of these Graft versus Host and Host versus Graft immune reactions are antigens encoded by the genes of the HLA (Human Leukocyte Antigen) system and that successful outcomes of bone marrow transplants are dependent on the sharing of HLA antigens by donor and recipient. Sibling donors who have inherited the same paternal and maternal HLA genes present in the recipient are HLA-identical and thus, optimal from this viewpoint. Patients lacking such HLA-identical sibling donors must receive transplants from more distant relatives or from unrelated donors. Because the HLA system includes several discrete genes each of which displays an extremely large number of antigenically different variants in the population, such distant relative-donor or unrelated-donor transplants must be expected to contain a variable number of HLA incompatibilities unless they are selected from among potential donors by identifying the specific variants present in each and choosing donors whose HLA antigens match those of the recipient. To perform this selection with significant probability of success, it is necessary to have access to large panels of potential donors whose HLA antigens are known. In the case of unrelated donor PB, this requires establishing a bank of frozen HLA-typed units collected from random placentas. Heretofore, the most widely accepted method for freezing PB consisted of adding to the whole PB unit an equal volume of a cryopreservative solution, with the double disadvantage that the volume of. each cryopreserved unit becomes very large and that a relatively large amount of possibly deleterious cryopreservative is eventually administered to the recipients of such PB units. Administration of cryoprotectant and hemoglobin from erythrocytes destroyed by using a freezing and thawing method designed to protect the stem and progenitor cells but not the erythrocytes may have toxic effects generally and especially on specific organs such as the kidney of the recipient. In addition, there is the logistical consequence that a large number of freezers would be needed to contain useful numbers of the large volume frozen units in reserve, with the attending increase in up-front and running costs. The applicants have developed a practical method that allows a substantial reduction of the volume of PB Units by eliminating the unneeded mature red blood cells and an equivalent volume of plasma. This submission describes this method and a specially designed set of plastic bags and connecting tubes intended to facilitate the accomplishment of the desired concentration of the needed stem cells and progenitor cells with minimal manipulation and risk of contamination. Essentially, this method will allow an experimental, time consuming laboratory process to become a routine procedure in blood banks.
The following submission reflects the state of the art of which applicant is aware insofar as these documents appear germane to the patent process. However, it is respectfully stipulated that none of these patents teach singly nor render obvious when considered in any conceivable combination the nexus of the instant invention as set forth hereinafter.
Pablo Rubinstein, Richard E. Rosenfield, John W. Adamson and Cladd E. Stevens (The Lindsley F. Kimball Research Institute of The New York Blood Center); Stored Placental Blood for Unrelated Bone Marrow Reconstitution; May, 1993; entire paper.
The therapeutic product of the present invention is advantageous, first, because it recovers all or almost all of the stem and progenitor cells of the original collection of PB in a small and uniform volume that requires minimal and predictable storage space, second, because it permits a consistent methodology for processing PB units which results in a routinely dependable product with less dependence on operator skill and third, because the potentially deleterious effects of the cryoprotectant and of the free hemoglobin are minimized.
One first aspect to the nature of the product improved according to the present invention involves the methodology by which the white blood cells (which include the hematopoietic stem and progenitor cells) are separated from the bulk of other components in the whole PB and the manner in which the viability of such white cells is preserved by avoiding exposure to bacterial and fungal contamination, potentially damaging chemical agents, excessive centrifugal forces and osmotic imbalances. Typically, bacterial and/or fungal contamination occurs when PB or white blood cell suspensions derived from PB are exposed to ambient air in the course of preparatory manipulations; chemical damage is possible when certain chemicals are used to lyse the accompanying red blood cells or to aggregate white cells; and physical damage follows the use of excessive centrifugal speed in separation of the cellular components of the blood according to their density, by centrifugal stratification. In addition, the method according to the present invention provides for avoidance of prolonged exposure of the separated white blood cells to cryopreservation solutions at room temperature, an exposure that results in decreased viability of the white blood cells and of the stem and progenitor cells contained therein because of osmotic imbalances and, possibly, other toxic effects of the intracellular cryoprotectants themselves.
Another aspect of the present invention involves the set of interconnected plastic containers (designated as bags). The set under the present invention permits a selective concentration of the white blood cells and of the stem and progenitor cells contained therein without reducing their normally high viability and freedom from contamination by infectious organisms from the environment. Whole PB is collected into a mother bag and is subsequently processed through a series of bags of appropriate chemical structure and physical shape and capacity culminating in storage of a separated fraction containing most of the white blood cells of the collected PB in liquid nitrogen at xe2x88x92196 C inside a specially constructed freezer bag. Intervening steps include the addition of substances that enhance the aggregability of red blood cells and the separation of components by transferring supernatants into connected satellite bags. A special bag and its connecting assembly permits the addition of measured amounts of cryoprotectant to the separated white blood cell concentrate. This connecting assembly allows the cryoprotectant to be added to the white cells at a precise, slow speed required to maintain optimal cell viability.
The bag which is to be used for freezing and storage includes a plurality of connected, but detachable, compartments for sequestration of the white blood cells into different discrete chambers. One chamber, the main compartment, is intended to keep the bulk of the white blood cells. A smaller compartment lends itself to the storage of a smaller fraction of the bag contents which may be separated from the main compartment without thawing, and extemporaneously detached from it for separate thawing and subsequent in vitro expansion of the hematopoietic stem and progenitor cell populations contained in the corresponding fraction of the white blood cells. A third and subsequent chamber contains very small aliquots of the white blood cell suspension that are intended to serve as detachable samples for testing the aptness of the unit to be transplanted or assessing its suitability as donor tissue for a specific recipient. The freezing bag also includes indicia on the outer surface of each of its detachable areas for identification of the specific unit that will be stored in it, to facilitate storage and retrieval from designated sectors of cryogenic storage depots. Means are also provided in an exterior surface of the freezer bag to facilitate the placing and removing of the freezer bag into and from, respectively, its assigned storage location by automated instrumentation.
Accordingly, it is a primary object of the present invention to provide the means for preparing PB derived hematopoietic stem and progenitor cells in a novel and therapeutically more useful form. The product becomes a bag containing a high concentration of white blood cells having a high degree of cell viability.
A further object of the present invention is to provide a novel and useful method for generating the therapeutic product according to the previous object.
A further object of the present invention is to provide an aseptic and interconnected bag set for use in conjunction with the method of developing the therapeutic product hereinabove.
A further object of the present invention is to provide a freezer storage bag configured to contain the therapeutic dose in a cryoprotected environment for protracted periods of time until needed for dosage.
A further object of the present invention is to provide a freezer bag as noted above provided with a plurality of compartments in which the therapeutic dose has been sequestered so that various aliquots can be strategically excised from the freezer bag for several purposes.
Viewed from a first vantage point, it is an object of the present invention to provide a system for developing placental stem cells, comprising in combination: a first blood bag adapted to receive blood from a placenta therewithin, means within the blood bag to prevent coagulation, reagent means removably coupled to the blood bag, means for separating supernatant from the first blood bag and into a white cell bag, means for separating white cells from plasma in the white cell bag, a plasma bag removably coupled to the white cell bag for receiving the expressed plasma from the white cell bag, cryoprotectant means operatively coupled to the white cell bag, and a stem cell freezing bag operatively coupled to the white cell bag for transferring contents from the white cell bag to the stem cell freezing bag.
Viewed from a second vantage point, it is an object of the present invention to provide a method for preparing concentrated and partially purified white blood cell suspensions containing placental stem cells, comprising the steps of: placing blood from a placenta into a first blood bag, preventing coagulation within the blood bag, coupling reagent means into the blood bag, centrifuging and separating white blood cell rich supernatant from the first blood bag and placing the supernatant into a white cell bag, separating white cells from plasma in the white cell bag, removably coupling a plasma bag to the white cell bag and expressing the plasma from the white cell bag into the plasma bag. Coupling cryoprotectant means to the white cell bag, transferring contents from the white cell bag to a stem cell freezing bag, and freezing the stem cell freezing bag with its contents follows.
Viewed from a third vantage point, it is an object of the present invention to provide a therapeutic product comprising at least 80% of the white blood cells (including stem and progenitor cells) with viability greater than 90% and fewer than 10% of the red blood cells in the original PB collection.
These and other objects will be made manifest when considering the following detailed specification when taken in conjunction with the appended drawing figures.